Background: Hepatitis B Virus (HBV) infection is a worldwide biomedical problem and an improved understanding of the assembly and structure of the virus may help develop new antiviral therapies as well as provide basic information on the structure of complex macromolecules. The HBV core gene codes for precore protein (pre-C) which is either partially processed to form a secreted non-particulate protein called e-antigen (HBeAg) or fully processed to produce core antigen (HBcAg). HBcAg is a 183-residue protein that encapsidate around a RNA-reverse transcriptase complex (HBV polymerase). HBcAg has been expressed in E.coli were it assembles in the bacterial cytoplasm into icosahedral capsids, which contain bound host nucleic acid. Deletion of the polybasic C-terminal 34 residues (protamine domain) also produces assembly competent protein. The capsids from C-terminal truncated protein (Cp149: residues 1-149) do not contain nucleic acid and their structure determined by cryo-electron microscopy and image analysis and by X-ray crystallography. Native HBeAg is also C-terminally truncated at position 149 and in addition contains a 10 residue N-terminal extension derived from partial processing of pre-C. Although the function and structure of HBeAg are unclear it is an important serological marker. Results: using surface plasmon resonance (Biacore), a kinetic-affinity map of a panel of monoclonal antibodies (mAbs) against HBV nucleocapsid proteins revealed a range of binding affinities. Monoclonal antibodies binding to the assembled and non-assembled forms of the capsids were identified. Some of the HBV nucleocapsid-antibody complexes were characterized further by cryo-electron microscopy (A. Steven). The results revealed a greater number of discontinuous epitopes than had been described previously. The findings help explain the immunological distinction between the assembled HBcAg and unassembled HBeAg antigen A review article was published summarizing these findings. Biophysical and structural studies on HBeAg are continuing using mutants with improved biophysical characteristics, especially solubility. We are preparing immune complexes of HBeAg with monoclonal antibodies to enhance crystal formation and subsequent structural studies using X-ray crystallography. Work is also continuing on our attempts to produce the HBV polymerase protein. We have also initiated studies on the HBV protein X, a key regulatory protein of the virus that is at the intersection of HBV infection, replication, pathogenesis, and possibly carcinogenesis. The protein has been expressed in E.coli and we are currently attempting to crystallize it for detailed structural determination. The surface glycoprotein of the HBV virus has also been expressed in yeast cells for potential structural determination. Summary: The Hepatitis B Virus (HBV) is the major worldwide cause of cancer. Although a vaccine is available, chronic HBV is often acquired in childhood. The HBV nucleocapsid plays an important structural role and metabolic role in the life cycle of the virus. An understanding of the molecular structure of the HBV nucleocapsid and other essential viral proteins including the polymerase and the X protein would allow targeted drug discovery with the aim of preventing the assembly and formation of the virus. A clearer understanding of the structural differences between the clinical HBV markers: HBcAg and HBeAg could result in better diagnostic tools.